Nano‐liposomal encapsulation of Artemisia aucheri phenolics as a potential phytobiotic against Campylobacter jejuni infection in mice

Abstract Background Artemisia aucheri contains antibacterial phenolic compounds. The current work was implemented to evaluate the effectiveness of a nanoliposome‐encapsulated phenolic‐rich fraction (PRF‐NLs), as a dietary phytobiotic derived from Artemisia aucheri's areal parts, on the inhibition of enteropathogenic Campylobacter jejuni (C. jejuni) infection in mice. Methods The phenolic‐rich fraction was loaded into the nanoliposome structure to obtain a nanometer‐scale size liposome with homogenous dispersion. Next, 40 white male balb/c mice were assigned to 4 treatment groups. The PRF‐NLs antibacterial potential was evaluated by evaluating the blood parameters, liver lipid peroxidation, and gene expression profiling in the mice challenged by C. jejuni infection. Results Mice infected by C. jejuni showed impairment in food intake, weight gain, liver function, ileum morphometric features, and ileum tissue inflammation. The diet of fortified food with the nonencapsulated and nanoliposome‐encapsulated phenolic compounds was found to improve these parameters at 10 mg TPC/kg BW/day concentration. Our data indicated that the nanoliposome‐encapsulated PRF was more effective in promoting the health parameters in mice as compared to nonencapsulated PRF. Conclusion It could be concluded that the liposomal encapsulation can promote the solubility, availability, and effectiveness of Artemisia aucheri phenolic compounds playing a key role as phytobiotic in mice intervened by enteropathogenic C. jejuni.

dependent on its solubility (Sur et al., 2019). Research has shown that nearly two-fifths of the available drugs in the market have poor water solubility, an issue that limits sufficient absorption, which leads to low therapeutic efficacy (Chaudhari & Akamanchi, 2019;Chen et al., 2019).
The liposome, as one of the best choices among the available transportation systems, has a spherical-shaped vesicle comprised of one or more phospholipid bilayers. The ability to encapsulate hydrophilic or hydrophobic drugs makes liposomes good candidates for drug delivery (Lee & Thompson, 2017;Olusanya et al., 2018). Nanotechnology has great attention nowadays and opens up a wide array of application in different filed including agriculture and medicine (Pramanik et al., 2020). Nanoliposomes are submicron bilayer lipid vesicles that encapsulate bioactive agents and increase drug performance by improving their solubility, stability, and bioavailability. The ability to target specific cells allows nanoliposomes to avoid unwanted interactions with nonspecific molecules (Bulbake et al., 2017;Wang et al., 2018).
Nanoliposomes have recently been used to design and construct delivery systems for the natural bioactive compounds, namely phenolic compounds. Phenolic compounds have undeniable biological impacts on human health (Esfanjani et al., 2018;Rafiee et al., 2017). They cast a major role in the suppression mechanisms (e.g., carcinogen inactivation, apoptosis induction, angiogenesis suppression, and antioxidation) of several types of cancer (Heleno et al., 2015). Moreover, the configuration and the quantity of the structural hydroxyl groups in their structure concern their antioxidant and antimicrobial capacity (Carocho & CFR Ferreira, 2013).
Also, several research articles have documented the polyphenols bioactivities.
Artemisia aucheri (Asteraceae), a perennial aromatic medicinal plant, is widely distributed throughout Asia, Europe, and America. The plant is known for its broad spectrum of bioactivity and natural bioactive compounds, videlicet polyphenols, flavonoids, terpenoids, and phenylpropanoids (Hussain et al., 2017;Kazemi, 2015). Several studies have underpinned the significance of A. aucheri's biological properties and therapeutic effects on human health. Therefore, the current work aimed to (a) produce a liposome-encapsulated A. aucheri polyphenol and (b) investigate its phytobiotic potential against Campylobacter jejuni (C. jejuni) infection in mice.

| Plant material and reagent
We purchased the A. aucheri fresh aerial parts from Mashhad herbal medicine market, Iran. The C. jejuni was purchased from the microbial culture collection of the Islamic Azad University of Mashhad, Iran. In this study, soybean lecithin was obtained from Sigma Aldrich. The RNA extraction and cDNA synthesis kits as well as SYBER Green master mix were all bought from Biofact company.
The other material and reagents not mentioned here were from Daejung.

| Fractionation
The areal parts of the A. aucheri plant were cleaned with sterile distilled water and dried in shadow for 2 weeks at room temperature.
The dried plant material was finely ground (powder form) using a grinder mill. Then, 100 g of the dried powder was extracted with 900 ml of aqueous methanol (80% (v/v)) and 100 ml of 6 M HCl using the reflux method for 2 h (Karimi et al., 2019). Then, the extract was filtered, and the filtrate was concentrated and evaporated at the temperature of 60°C by using a rotary evaporator (Buchi). In the next step, the extract was fractionated using a separating funnel and different solvents including hexane, chloroform, ethyl acetate, n-butanol, and water-based as described earlier Oskoueian et al. (2020). Upon fractionation, the supernatant was filtered and concentrated using a vacuumed rotary evaporator. The total phenolic compound (TPC) evaluation of each fraction was carried out by adding 0.1 ml of the extract, 2.5 ml of Folin-Ciocalteu reagent (1:10 v/v), and 2 ml of 7.5% sodium carbonate into a test tube covered with aluminum foil. The test tubes were vortexed and the absorbance was measured at 765 nm as described earlier by Oskoueian et al. (2020).
The results were expressed as milligrams of gallic acid equivalents (GAE) per gram of dry weight. The fraction containing the highest phenolic content is named a phenolic-rich fraction (PRF).

| Preparation of nano-liposomal carriers
To prepare the nanoliposomes, 4 g of lecithin was added to 196 g of 80°C hot water and stirred for 2 h. In the next step, the PRF was added and stirred for 2 additional hours to attain 2000 ppm as the final concentration. Ultimately, the solution was sonicated for 6 min; the acquired nanoliposome-loaded PRF was synthesized for further characterization (Beyrami et al., 2020).

| Characterization of nanoliposomes
After diluting the nanoliposomes-loaded PRF in water (1:20), the dynamic light scattering (DLS) protocol was used to determine the zeta potential of the particles. Field Emission Scanning Electron Microscopy (FESEM) was additionally employed to determine the nanoliposomes' size dimensions. Malvern Zetasizer Nano ZS was recruited to analyze the measurements three times. and solvent B (acetonitrile). Before injection, we equilibrated the column with 85% of solvent A and 15% of solvent B for 15 min. After 50 min, the ratio of solvent B was increased to 85%. At minute 55, the ratio of solvent B was declined to 15%; this ratio was maintained for 1 h to run the subsequent analysis with a flow rate of 0.6 ml/ min. As described previously, gallic acid, syringic acid, vanillic acid, salicylic acid, caffeic acid, pyrogallol, catechin, cinnamic acid, ellagic acid, naringin, chrysin, and ferulic acid were considered phenolic standards (Karimi et al., 2018).

| In vivo experiment
Forty (20-25 g) white male Balb/c mice were purchased from the Razi Vaccine and Serum Research Institute of Mashhad, Iran. All mice were kept in individual cages at 23 ± 1°C and 58% ± 10% humidity; they were exposed to 12-h light/dark periods for 7 days for adaptation to laboratory conditions. Ten mice were assigned to each group in 4 groups overall. The mice had access to tap water and were fed from the standard pellet diet produced by Javaneh Khorasan, Mashhad, Iran. The experimental treatments were: Animals received the treatments for 4 weeks. All samples were gavaged orally using 10 8 cfu of C. jejuni on day 21. We monitored the mice on daily basis for general health and the quantity of diet. On day 28 (the end of the experiment), pentobarbital-HCL (50 mg/kg, i.p.) was used to euthanize the mice. Immediately, the blood, liver, and ileum samples were excluded and liver enzyme analysis, lipid peroxidation assay, gene expression analysis, and the morphometric analysis of the ileum were evaluated. The mice were weighed on altered occasions throughout the experiment: the beginning, the middle, and the end of the process. All animal experiments were implemented following the research ethics approved by the Islamic Azad University of Mashhad.

| Liver enzymes and lipid peroxidation assay
The liver enzymes, such as SGOT, SGPT, and ALP, were determined in the serum. The liver's lipid peroxidation was examined based on Shafaei et al. (2020). The samples' absorbance was read at 532 nm; the output was presented as a percentage relative to the control.

| Histopathology analysis
The mice were euthanized, and their livers, kidneys, and ileum were washed in the normal saline. Eventually, the separated organs were preserved in 10% buffered formalin (in 0.1 M sodium phosphate buffer, pH7) and paraffinized, sliced for staining following Shafaei et al. (2020) hematoxylin/eosin protocol.

| Validation of gene expression
To evaluate the molecular mechanism of action of PRF nanoliposomes in promoting the health status of the mice, we determined the expressions of fundamental biomarker genes, namely COX2, iNOS, SOD, and GPx. Liquid nitrogen was used to freeze the ileum tissues immediately upon sampling. The frozen tissues were then crushed in mortar and pestle in the presence of liquid nitrogen to prepare for RNA extraction using an RNA extraction kit. Next, we synthesized the cDNA libraries entirely using a cDNA synthesis kit. Furthermore, the sets of primer sequences were used to investigate the expression level of key genes as well as the beta-actin as the housekeeping gene. Noteworthy, we employed the SYBR Green PCR Master Mix in a comparative qPCR (Roche Diagnostics). The (target) genes were amplified accordingly at Total bacteria cggcaacgagcgcaaccc ccattgtagcacgtgtgtagcc Denman and McSweeney (2006) reference gene) and the relevant genes in the control group (Kathirvel et al., 2010). See Table 1 for the primers' characteristics.

| Analysis of ileum microbial population
The qPCR was performed to characterize the population of C. jejuni in the ileum. Briefly, to extract the DNA from 1 ml of the ileum digesta, we used the QIAamp DNA Stool extraction kit (QIAGEN). Table 2 illustrates the primers used in this study. In addition, we adopted 2 −∆∆Ct method to analyze the data from the qPCR to identify the fold changes in the C. jejuni population (Feng et al., 2010). The 2 −∆∆Ct displays the difference between the ΔCt values at the pre-and postbacterial challenge. The cycle threshold (also, Ct) is known to be the point where the fluorescence rises above the background. The qPCR system software was recruited to determine the Ct values according to the threshold line tuned manually above the noninformative fluorescent data.

| Procedures of data analysis
All experiments were performed in triplicates, and statistical analysis of data was expressed as Means ± standard deviations (SD). We ran Duncan's Multiple Range Test to determine the significance of the means differences (p < .05).

| Fractionation and identification of phenolic compounds
The TPC found within different fractions of A. aucheri indicated that the phenolic compounds varied from 4.7 ± 2.02 to 52.4 ± 1.45 mg gallic acid equivalent (GAE)/100 g dried fraction. Whereas the ethyl acetate fraction (52.4 ± 1.45 mg GAE/g DM) was found to contain the highest amount of phenolic content, the hexane fraction showed the lowest amount (4.7 ± 2.02 mg GAE/g DM). Several early studies indicated that the phenolic compounds are moderately polar compounds; that is why they tend to accumulate in the fraction of medium polarity such as ethyl acetate (Kaur et al., 2008;Olatunji et al., 2017). The ethyl acetate fraction, as a PRF, was selected for the encapsulation process and further evaluations. In line with our findings, previous studies (Abdelwahab et al., 2010;Kaur et al., 2008) reported that while various polarity solvents were used to extract phenolic compounds, the highest TPC was found in the ethyl acetate fraction.

| Size, zeta potential, and morphology of PRF-NLs
Characteristics such as size, zeta potential, and morphology of the nanoliposomes are considered important parameters for stability, bioavailability, and the release behavior of nanoparticles. As demonstrated in Table 3, the PRF encapsulated in liposome carrier had a nanometer size (170.3 ± 6.89 nm); hence, it was named phenolic-rich fraction-loaded nanoliposome (PRF-NLs). The PRF-NLs exhibited a negative surface charge (−21.04 mV) (see Figure 1), indicating low to moderate stability based on Kumar and Dixit's (2017) classification.
Furthermore, the PDI value (0.258) showed that PRF-NLs had high homogeneity and narrow particle size distribution. Figure 2 illustrates the PRF-NLs morphology, demonstrating its spherical shape and distribution.

| TPC and HPLC analysis of PRF-NLs
According to our data, the TPC of the PRF-NLs was 18.7 ± 2.18 mg GAE/g DM. Phenolic compounds profiling by HPLC unveiled

F I G U R E 1
The zeta potential of liposomes containing Artemisia aucheri phenolics-rich fraction various types of natural phenolic compounds (see Table 4).

| Mice trial
According to Table 5, mice with C. jejuni infection (T2) demonstrated a considerable decrease (p < .05) in the average daily weight gain and dietary intake in comparison to their un-infected counterparts (T1). Supplementation of the PRF to the infected (with C. jejuni) groups (T3 and T4), in the form of nonencapsulated and nanoliposome-encapsulated, significantly improved (p < .05) these parameters. These results revealed that supplementing liposome-encapsulated phenolic compounds was more effective in improving daily weight gain and food intake in the infected mice than those supplemented with nonencapsulated.
Such difference could be ascribed to liposomal encapsulation, which enhanced the solubility, bioavailability, and the effectiveness of the antioxidant and antibacterial activity of the bioactive compounds in the PRF.

| Liver enzyme and lipid peroxidation analysis
According to Table 6    Average daily feed intake (g) 3.1 a 2.5 c 2.7 bc 2.9 b 0.12

| Histological and morphometric analysis
Note: Different letters in the same row indicate significant difference (p < .05). The analysis was performed in triplicates.

TA B L E 5
The body weight changes and feed intake analysis Note: Different letters in the same row indicate significant difference (p < .05).
*Changes relative to control.

F I G U R E 4
Relative quantification of C. jejuni population in ileum digesta. T1, normal diet; T2, normal diet +infected by C. jejuni on day 21; T3, normal diet enriched by a nonencapsulated phenolic rich fraction (10 mg TPC/kg BW/day) + infected by C. jejuni on day 21; T4, normal diet enriched by a nanoliposomeencapsulated phenolic rich fraction (10 mg TPC/kg BW/day) + infected by C. jejuni on day 21

| CON CLUS ION
According to the obtained results, nonencapsulated and nanoliposome-encapsulated PRFs of A. aucheri, with a concentration of 10 mg TPC/kg BW/day, could inhibit the pathogen population and improve the mice health parameters when challenged by enteropathogenic C. jejuni. The delivery of nanoliposome-encapsulated phenolic complexes was found more active in improving the health parameters than the nonencapsulated phenolic compounds. Thus, it could be concluded that liposomal encapsulation may promote the solubility, availability, and effectiveness of A. aucheri phenolic compounds. As a phytobiotic, A. aucheri phenolics are found to cast a prominent role in the inhibition of enteropathogenic C. jejuni infection in mice. The isolation and encapsulation of phenolics individually and evaluation of their phytobiotic potential against C. jejuni are recommended for the future works.

CO N FLI C T O F I NTE R E S T
The authors have declared that there are no competing interests.

E TH I C A L A PPROVA L
All protocols to conduct an in vivo study were described in compliance with ARRIVE guidelines. All animal investigations were carried out based on the ethical principles approved by the Islamic Azad University of Mashhad, Iran (code no: IR.IAU.MSHD.